Biomolecules specifically binding to target substances or low molecular compounds whose target molecules are biomolecules have been expected to be used as candidate substances for pharmaceutical drugs which exert effective physiological activities in vivo on the basis of their specific binding functions to target substances or for target substance-capturing bodies of biosensors.
An example of the biopolymers as described above can include antibodies. The antibody is one of proteins that function in the self-defense mechanisms of animals through which various foreign substances invading their body fluids are detoxicated by the immune systems. In other words, the immune system recognizes a variety of structures on the surface of the foreign substance and produces antibodies specifically binding thereto. As a result, the specific binding of the antibody to the foreign substance detoxicates the foreign substance through the in-vivo immune system. To effectively exert this mechanism, antibodies possess molecular diversity (the number of antibodies with different amino acid sequences for binding to various foreign substances), and the number of antibodies per individual animal is estimated to be 107 to 108. Their specificity in antigen recognition, high antigen-binding ability and molecular diversity account for expectations placed on the use of the antibodies as candidate substances for pharmaceutical drugs or as target substance-capturing bodies.
An antibody has a structure formed by two long and two short polypeptide chains. The long polypeptide chains and the short polypeptide chains are called heavy chains and light chains, respectively. These heavy and light chains individually have variable and constant regions. The light chain is a polypeptide chain composed of two domains, one variable region (VL) and one constant region (CL). The heavy chain is a polypeptide chain composed of four domains, one variable region (VH) and three constant regions (CH1 to CH3).
Each domain of the antibody assumes a tubular structure consisting of approximately 110 amino acids and forms a very stable structure where layers are formed by β-sheets arranged in an antiparallel orientation and are further bound with each other through SS-bond.
The binding of the antibody to various antigen species is known to result from the diversity of amino acid sequences of three complementarity determining region (CDR) retained in each variable region (VH or VL). These three CDRs residing in each of VH and VL are partitioned by framework regions and allow for more highly specific molecular recognition by recognizing the spatial arrangement of a substance to be recognized. The diversity of CDR is generated by DNA reorganization occurring in the antibody gene loci when bone marrow stem cells are differentiated into B lymphocytes, antibody-producing cells. This diversity is known to be produced by causing the DNA reorganization in portions composed of VH, D and JH gene fragments in the heavy chain and in portions composed of Vλ or Vκ gene fragments or Jλ or Jκ gene fragments in the light chain. These genetic recombination processes allow for the molecular diversity of the antibody.
Such antibodies capable of binding to particular substances have conventionally been produced in artificial manners utilizing the antibody production mechanisms in the immune systems of animals as described above and have been used in various industrial fields. One example of the production method thereof includes a method involving immunizing animals (e.g., rabbits, goats and mice) to be immunized with antigen substances of interest together with adjuvants at certain intervals and collecting antibodies produced in their sera. The antibodies thus obtained are a mixture of plural antibodies that recognize various structures present on the surfaces of the antigen substances used in the immunization. The sera containing plural antibodies binding to single antigens as described above are called polyclonal antibodies.
On the other hand, the DNA reorganization occurs independently in each B cell. Therefore, one B cell produces only one type of antibody. To obtain single antibodies, a method involving fusing B cells producing particular antibodies with established tumor cells to produce hybridoma cells has been established. The single antibodies produced from such hybridomas are called monoclonal antibodies.
Antibody fragments Fab, Fab′ and F(ab′)2 obtained by treating the antibodies as described above with a certain kind of proteolytic enzyme are known to have binding ability to the same antigen as those against their parent antibodies and known to be sufficiently available as target substance-capturing bodies.
As described above, such antibodies or antibody fragments are widely available as target substance-capturing bodies adapted for target molecules in biosensors. In this case, the antibodies or antibody fragments are generally immobilized for use on a substrate. A method used for immobilizing the antibodies or antibody fragments are generally selected from physical adsorption and chemical crosslinking methods. In the immobilizing method using physical adsorption, a site of the protein involved in the adsorption cannot be selected arbitrarily when physically adsorbed onto the substrate. Alternatively, in the immobilizing method using chemical bond caused by crosslinking reaction, a functional group of the protein involved in reaction with a crosslinking agent cannot be determined arbitrarily in most cases. Furthermore, when there exist plural reactable functional groups, selectivity among them is exceedingly low. In binding to the substrate through physical adsorption or through chemical bond caused by crosslinking reaction, a site of the protein involved in the binding is generally selected at random. Therefore, if a site directly or indirectly involved in the target substance-binding ability of a protein is identical or overlaps with a site involved in binding onto substrate surface, the target substance-binding ability of the protein might be reduced remarkably.
Moreover, studies have been conducted, which apply Fab and Fab′ fragments containing heavy chain variable regions (VH) and light chain variable regions (VL) that are antibody recognition domains or containing constant regions CH1 and CL for stabilizing them more highly, camel heavy-chain antibody variable regions (VHH), VH and VL. In these studies, a single chain antibody (scFv) is produced in a genetic engineering manner by fusing VH, VL, and so on via amino acids called linkers, and applied as a target substance-capturing body.
For a method for immobilizing such antibody fragments onto a substrate, their features of being able to be produced in a genetic engineering manner have been exploited to study a method involving fusing substrate-affinity peptides or biological compounds (e.g., enzymes) with affinity for compounds immobilized on the substrate into the antibody fragments in a genetic engineering manner. According to this method, such peptides or biological compounds can be selected and fused with the amino terminus (N terminus) or carboxy terminus (C terminus) of the produced antibody fragment molecules so as not to affect their desired antigen-binding ability. Therefore, the antibody fragments bound on the substrate can be expected to be oriented to some extent.
Examples of the substrate-affinity peptides include His tag composed of plural (usually five or more) consecutive histidine residues bonded together. If using a recombinant protein fused with this His-tag, it is possible to arrange desired target substance-capturing bodies on the substrate by applying coating capable of maintaining Ni ions to the substrate surface and utilizing the electrostatic binding between the Ni ions and the His tag.
Anal. Chem. 2004, 76, pp 5713-5720 has disclosed the use of a fusion protein comprising cutinase fused with the N terminus of scFv or the C terminus of VHH against hen egg lysozyme (HEL). This fusion protein is immobilized onto a substrate as follows: at first, SAM layers consisting of triethylene glycol sulfide displaying a suicide substrate for cutinase are formed on a gold substrate and thus the antibody fragment of interest is immobilized onto the substrate via the irreversible binding between the suicide substrate and the cutinase. This document has also disclosed that the antibody fragment immobilized by this method exhibits desired binding ability.
A method for obtaining (producing) the antibodies or antibody fragments capable of being produced in a genetic engineering manner as described above is expected as follows: production methods or systems requiring low cost in total are expected to be adopted in consideration of investment in production facilities using prokaryotes typified by E. coli as hosts, production control during the operation of the production facilities, etc. However, it is difficult to produce, in the prokaryotes, proteins derived from higher organisms including humans as active proteins that maintain desired functions. In many cases, general methods for this purpose have not been established.
A method selected for producing the antibody fragments typified by scFv and Fab in E. coli is a method involving arranging a secretion signal such as pelB at the N terminus and allowing E. coli to secrete the active antibody fragments into the periplasm or a culture supernatant by utilizing the mechanism of inner membrane transport. However, in such a method, antibody fragments cannot be obtained as secreted proteins for some types of antibodies of interest. For example, the desired antibody fragments are sometimes produced as an aggregate of insoluble fractions into the bacterial cell and are not secreted. In this case, the step of solubilizing the obtained aggregate with a denaturant such as guanidine hydrochloride and then refolding the protein structure into an active structure by a dilution or stepwise dialysis method is required and is operationally complicated. Moreover, active protein yields sometimes fall short of acceptable levels.
On the other hand, a method involving fusing the antibody fragments obtained in a genetic engineering manner with secretory proteins to efficiently obtain the antibody fragments as fusion proteins has been known. U.S. Pat. No. 5,969,108 has disclosed a technique for using the antibody fragment as described above as a phage antibody having a structure where the antibody fragment is fused with a coat protein of a phage, particularly a filamentous phage, and expressed and displayed on its surface.
In the production of phages displaying antibodies on their coat protein surfaces, a method involving selecting antibody fragment clones have been utilized as disclosed in the pamphlets of International Publication No. WO088/06630 and International Publication No. WO092/15677 in addition to U.S. Pat. No. 5,969,108 described above.
However, these documents have merely disclosed a method (antibody display method) for fusing an antibody of interest with a phage minor coat protein pIII (hereinafter, pIII) and has not specifically mentioned an antibody display method for other coat proteins. On the other hand, the pamphlet of WO092/15679 has disclosed a specific method for displaying a desired protein on a phage major coat protein pVIII (hereinafter, pVIII).
This document has gained the findings that an antibody protein displayed on pIII causes irreversible reaction with a target substance, and has released a technique for displaying a desired protein on pVIII and effects thereof.
However, all of the documents described above have merely disclosed the technique for displaying an antibody or desired protein on pIII and pVIII.
Descriptions suggesting the immobilization of these antibody fragment-fused phages on a particular substrate or the use of the immobilized phages rendered functional as sensing devices are not found in any of these documents.
On the other hand, Chemistry & Biology, Vol. 11, pp 1081-1091 has disclosed that labeled streptavidin can be detected on a cell.
More specifically, two different peptide chains are displayed on different coat proteins (pIII and pVIII) of a filamentous phage. The peptide chain (RGD-4C: CDCRGDCFC) displayed on the pIII is bound with a target substance (integrin) immobilized on a cell.
As a result, the phage is immobilized on the cell (KS1767) while the streptavidin-binding peptide (R5C2: ANRLCHPQFPCTSHE) is displayed on the pVIII of the phage. According to this document, the labeled streptavidin can thereby be detected on the cell. The document has also disclosed that the phage is bound with a substrate coated with streptavidin to detect the cell.
However, Chemistry & Biology, Vol. 11, pp 1081-1091 has demonstrated that difference in binding with the KS1767 cell is small between the RGD-4/R5C2 peptide-displaying phage and the R5C2-displaying phage. This indicates the low target substance-recognizing (binding) ability of the displayed RGD-4 peptide. Thus, this technique leaves room for improvements from the viewpoint of its use as a biosensing device that specifically detects a particular substance on a substrate.
The most important technique for producing and industrially using an excellent biosensing device is to produce binding molecules (e.g., antibodies) highly specific to target substances at high yields and immobilize the molecules on a substrate or the like with their activities maintained.